Cell differentiation at the molecular level is still not well understood. The long-term objective of this proposal is to elucidate the molecular mechanisms involved during differentiation of the parasitic protozoan Leishmania mexicana amazonensis. Regulation of tubulin gene expression has been chosen for an initial study, because leishmanias have a motile, flagellated promastigote stage and a nonmotile, intracellular amastigote stage in their life cycle, and the two stages, as well as transformation between stages, can be maintained under laboratory conditions. Leishmanias possess a variety of functionally distinct microtubules, including flagellar, subpellicular and nuclear spindle types. With the hybridoma technique, monoclonal antibodies will be generated; and antibodies against specific components of the leishmanial cytoskeleton, or against specific types of microtubules, will be selected, and antigenic changes during amastigote-to-promastigote transformation in vitro will be identified. The aim is to dissect the cytoskeletal elements with monoclonal antibodies because the leishmanial microtubular system can be viewed as a potential target structure for chemotherapeutic attack. Expression of tubulin genes during leishmanial differentiation will also be analyzed at the nucleic acid level. Tubulin specific cDNA clones will be selected from leishmanial cDNA libraries using probes containing Chlamydomonas or chicken tubulin sequences. Restriction maps of cloned tubulin inserts will be used to characterize the different stage-specific tubulin RNAs being expressed by the amastigotes and the promastigotes. S1 nucleases protection experiments will be performed in order to compare the tubuline mRNAs between the two developmental stages. Restriction fragments of tubulin coding and flanking sequences will be used to investigate both the tubuline gene organization in the leishmanial genome, and the expression of tubulin RNAs during leishmanial differentiation. The aim is to study the leishmanial tubulin system as a model for the regulation of gene expression during cell differentiation.